The present invention relates to the fields of molecular biology, chemistry and nucleic acid hybridization. In certain embodiments, the present invention provides methods and compositions that are useful for detecting differences between nucleic acids.
Breakthroughs in genetics have identified numerous traits that have been associated with diseases. Such traits could be used to accelerate the prevention or treatment of the diseases. For some diseases, a single genetic marker is sufficient to indicate a predisposition for a disease. Detection of the marker can thus indicate an individual at risk for the disease. However, for many diseases, multiple genetic markers interact to generate complex genetic traits that are associated with the diseases. For such diseases, detection of multiple genetic markers might be needed to for the treatment or prevention of the disease. Methods of rapidly and accurately detecting such genetic markers are needed to improve the treatment or prevention of diseases that can be associated with genetic markers.
Many such genetic markers are single-nucleotide polymorphisms (SNPs). Such SNPs are distributed throughout the genome at frequency of about 1 per 1,000 base pairs. Several hundred thousand of these markers are now available in public databases. These databases should facilitate the identification of genetic markers associated with simple and complex diseases. However, efficient methods to rapidly identify and/or detect SNPs are needed to utilize such genetic markers for effective treatment or prevention of the diseases.
Conventional methods have been used for the detection of SNPs and other genetic markers. Traditional methods include direct sequencing of polynucleotides and direct measurement of restriction fragment length polymorphisms. In addition, methods based on the hybridization of probes to genetic markers have been used. Such methods include oligonucleotide chips, polymerase chain reaction amplification of genetic markers and other such techniques.
However, such conventional techniques often suffer from poor accuracy, high cost or low throughput. For example, current hybridization-based genotyping methods such as SNP-chip or micro-array often lack sufficient sensitivity and/or accuracy to detect many SNPs simultaneously with a uniform set of conditions. A polynucleotide comprising one version of an SNP is often capable of hybridizing to a polynucleotide comprising a second version of the same SNP when assayed according to conventional techniques. Although hybridization is stronger between two perfectly complementary polynucleotides, single base-pair differences are often not sufficient to detect many SNPs simultaneously with the same set of conditions required for SNP-chip or micro-array.
The need thus remains for a less costly, more accurate method to detect the presence or absence of sequence differences between polynucleotide samples, for example, at many different SNP positions with very high throughput.
Accordingly, the present invention provides methods for detecting the presence or absence of a difference between two related nucleic acid sequences. The methods achieve sensitivities great enough to detect the presence of any difference between the nucleic acids, even single nucleotide polymorphisms.
In one aspect, the present invention provides methods of detecting a difference between two nucleic acids. In the methods, the nucleic acids are contacted under conditions in which they are capable of forming a four-way complex. A four-way complex is a macromolecular structure that comprises both nucleic acids in double stranded form. Typically, a four-way complex comprises a Holliday junction. A Holliday junction is known to those of skill in the art as the branch point in a complex of two related (often identical) double stranded nucleic acids. If the nucleic acids share identical sequences and the sequence identity extends to the ends of the nucleic acids, the four-way complex is capable of undergoing branch migration under the appropriate conditions resulting in resolution into two double stranded sequences. Significantly, if sequence identity and complementarity does not extend to the ends of the nucleic acids, migration of the four-way complex can halt at or near a site where the sequences are not identical or complementary.
The conditions under which the nucleic acids are contacted are chosen so that the four-way complex is capable of allele-specific four-way complex migration. Such conditions are known to those of skill in the art and include those under which migration of a four-way complex can proceed along the strands of the nucleic acids that comprise identical or complementary sequences. Typically, conditions are chosen such that migration will proceed to completion only if the two nucleic acids are identical in sequence. Thus, if there is no difference between the nucleic acids, migration can proceed to completion thereby resolving the four-way complex to yield two double stranded nucleic acid products. If there is a difference in sequence between the two nucleic acids, one or more base mismatches can form that can be capable of impeding four-way complex migration resulting in a stabilized four-way complex if the mismatches pose a sufficient energy barrier to migration. Detection of the stabilized four-way complex indicates a difference between the sequences of the nucleic acids.
In certain embodiments of the invention, mutations can be introduced into one or both nucleic acids to promote allele-specific four-way complex migration. Such mutations are typically near the site of a polymorphism and do not impede four-way complex migration unless the nucleic acids differ at the site of the polymorphism. Such mutations are described in detail in copending U.S. application Ser. No. 10/071,302, the contents of which are hereby incorporated by reference in their entirety.
In order to detect any stabilized four-way complexes, the nucleic acids are contacted with a detection molecule in a solution comprising a tracer molecule or tracer molecules. The detection molecule can be any molecule that is capable of selectively binding a four-way complex of nucleic acids such as a Holliday junction. Suitable detection molecules are known to those of skill in the art and include, but are not limited to RuvA, RuvC, RuvB, RusA, RuvG, and mutants, analogs or fragments thereof. Binding of the detection molecule by a stable four-way complex of the nucleic acids indicates a difference between the nucleic acids. This binding can be detected with the tracer molecules.
The tracer molecule can be used to detect the binding of the detection molecule to the stable four-way complex of the nucleic acids. The tracer molecule can be any molecule capable of selectively binding the detection molecule. When co-existing in solution, the tracer molecule can compete with the four-way complex for binding to the detection molecule. Preferably, the tracer molecule comprises one or more oligonucleotides that are capable of forming a stable or immobile four-way complex. Significantly, the tracer molecule also comprises a detectable label. The detectable label should be capable of generating a detectable signal that is sensitive to binding of the tracer molecule by the detection molecule. In other words, the detectable signal upon binding by the detection molecule should be distinguishable from the detectable signal in the absence of binding by the detection molecule. The detectable signal can be detected by methods known to those of skill in the art for detecting the signal.
Binding of the four-way complex of nucleic acids by the detection molecule is indicated by detecting the extent of the binding of the tracer molecule by the detection molecule. If the four-way complex of nucleic acids is stable, the four-way complex should compete with the tracer molecule for binding by the detection molecule. As such, the level of the detectable signal generated upon binding of the tracer molecule to the detection molecule will be determined by the existence/quantity of the co-existing competitive stable four-way complex. As a result, the level of the detectable signal indicates a stable four-way complex and thus the existence or absence of a difference between the nucleic acids.
The methods and compositions of the invention can be used in any application for which the detection of differences between nucleic acids is useful. Such applications include genotyping, SNP identification, SNP scoring, nucleic acid sequencing, and so forth. The methods and compositions of the invention provide sensitive and efficient methods of detecting any difference between two nucleic acids. Moreover, the methods and compositions of the invention can also be used for the detection/quantification of any nucleic acids of interest, including both DNA and RNA.